US20040235902A1 - Fluorescent probes for zinc - Google Patents

Fluorescent probes for zinc Download PDF

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US20040235902A1
US20040235902A1 US10/479,517 US47951704A US2004235902A1 US 20040235902 A1 US20040235902 A1 US 20040235902A1 US 47951704 A US47951704 A US 47951704A US 2004235902 A1 US2004235902 A1 US 2004235902A1
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group
compound
salt
zinc
hydrogen atom
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Tetsuo Nagano
Kazuya Kikuchi
Tomoya Hirano
Satoko Maruyama
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Daiichi Pure Chemicals Co Ltd
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Assigned to DAIICHI PURE CHEMICALS CO., LTD., TETSUO NAGANO reassignment DAIICHI PURE CHEMICALS CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HIRANO, TOMOYA, KIKUCHI, KAZUYA, MARUYAMA, SATOKO, NAGANO, TETSUO
Publication of US20040235902A1 publication Critical patent/US20040235902A1/en
Priority to US12/058,289 priority Critical patent/US20080261314A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1011—Condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/18—Metal complexes
    • C09K2211/188—Metal complexes of other metals not provided for in one of the previous groups

Definitions

  • the present invention relates to a fluorescent probe for specifically trapping a zinc ion.
  • Zinc is an essential metallic element that is present in the human body in the largest amount next to iron. Most zinc ions in cells strongly couple to proteins and are involved in the maintenance of structure or in the expression of function of the protein. Various reports have been also made on the physiological role of free zinc ions, which are present in the cell in a very small quantity (generally at a level of ⁇ M or lower). In particular, zinc ions are considered to be significantly involved in one type of cell death, i.e., apoptosis, and it is reported that zinc ions accelerate senile plaque formation in Alzheimer's disease.
  • a compound (a fluorescent probe for zinc), which specifically traps a zinc ion to form a complex and emits fluorescence upon the formation of the complex, has been conventionally used to measure zinc ions in tissue.
  • TSQ Redyes, J. G., et al., Biol. Res., 27, 49, 1994
  • Zinquin ethyl ester Tinylaminoethylcyclen
  • Dansylaminoethylcyclen Keike, T. et al., J. Am. Chem.
  • the present inventors As a highly sensitive fluorescent probe for zinc which has overcome defects of the conventional fluorescent probes such as TSQ, the present inventors have provided a probe which has a cyclic amine or a polyamine as a substituent, and traps zinc ions to emit intensive fluorescence with long wavelength excitation light (Japanese Patent Unexamined Publication No. 2000-239272). The present inventors have also provided a probe which quickly reacts with a zinc ion to form a fluorescent complex, enabling a measurement of zinc in organisms with excellent accuracy and high sensitivity (J. Am. Chem. Soc., 2000, 122, 12399-12400).
  • concentrations of the fluorescent probe introduced into cells may sometimes vary depending on types of cells. There are also many factors which influence measurements, such as possibilities that thickness of cell membranes may cause differences of fluorescence intensities in area to be measured, and fluorescent probes may localize in highly hydrophobic moiety such as membranes.
  • the ratio measurement method As a method that can reduce measurement errors caused by these factors to achieve a precise quantitative measurement, the ratio measurement method has been developed and used (Kawanishi Y., et al., Angew. Chem. Int. Ed., 39(19), 3438, 2000).
  • the method comprises a step of measuring fluorescence intensities at different two wavelengths in a fluorescence spectrum or an excitation spectrum to detect the ratio of the intensities.
  • the influences of concentrations of a fluorescent probe, per se, and the excitation light intensities are negligible, and measurement errors that are derived from localizations, changes in concentration, or discolorations of a fluorescent probe itself can also be eliminated.
  • Fura 2 (1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid, pentapotassium salt: Dojindo Laboratories 21st edition/general catalog, 137-138, published on Apr. 20, 1998, DOJINDO LABORATORIES Corporation) has been practically used.
  • the compound has a feature that a peak of an excitation wavelength shifts to shorter wavelength by binding to a calcium ion.
  • the fluorescence intensity increases with increase of the concentration of calcium ions
  • the fluorescence intensity reduces with increase of the concentration of calcium ions. Therefore, by excitations of the compound at two suitable wavelengths, and by calculation of the ratio of the fluorescence intensities at the two wavelengths, calcium ions can be precisely measured irrespective of probe concentration, light source intensity, the size of cells, and the like.
  • the inventors of the present invention conducted various studies to achieve the foregoing objects. As a result, they found that a compound represented by the following general formula (I) can specifically trap a zinc ion, and give a remarkable wavelength shift of a peak in an excitation spectrum, and by using said compound, a zinc ion can be measured with excellent accuracy by the ratio measurement method.
  • the present invention was achieved on the basis of these findings.
  • the present invention thus provides a compound represented by the following general formula (I) or a salt thereof:
  • R 1 represents hydrogen atom, an alkyl group, an alkoxy group, or hydroxy group
  • R 2 represents a group represented by the following formula (A):
  • X 1 , X 2 , X 3 , and X 4 each independently represents hydrogen atom, an alkyl group, or 2-pyridylmethyl group, and m and n each independently represents 0 or 1);
  • Y represents a single bond or —CO—;
  • R 3 represents a carboxy-substituted aryl group, a carboxy-substituted heteroaryl group, benzothiazol-2-yl group, or 5-oxo-2-thioxo-4-imidazolyzinylidenmethyl group].
  • the aforementioned compound or a salt thereof wherein m is 0 or 1 and n is 0; the aforementioned compound or a salt thereof wherein both of X 1 and X 2 are 2-pyridylmethyl groups, and when m is 1, X 3 is hydrogen atom; the aforementioned compound or a salt thereof wherein Y is a single bond; the aforementioned compound or a salt thereof wherein R 1 is methoxy group; the aforementioned compound or a salt thereof wherein R 3 is a carboxy-substituted aryl group or a carboxy-substituted heteroaryl group; the aforementioned compound or a salt thereof wherein the carboxyl group substituting on the aryl ring or the heteroaryl ring has a protective group; and the aforementioned compound or a salt thereof wherein the carboxyl group having a protective group is methoxycarbonyl group or acetoxymethyloxycarbonyl group.
  • X 1 and X 2 are 2-pyridylmethyl groups
  • X 3 is hydrogen atom
  • Y is a single bond
  • R 1 is methoxy group
  • R 3 is p-carboxyphenyl group
  • the present invention provides a fluorescent probe for zinc which comprises the aforementioned compound or a salt thereof; a zinc complex which is formed with the aforementioned compound or a salt thereof and a zinc ion; and an agent for measuring zinc ions which comprises the aforementioned compound or a salt thereof.
  • FIG. 1 shows changes in excitation spectra of the compound of the present invention (Compound (11)) depending on concentration of zinc ions.
  • FIG. 2 shows the result of the ratiometric measurement of zinc ions and other metal ions by using Compound (11).
  • (A) shows a change in the ratio by addition of zinc ions
  • (B) shows changes in the ratio by addition of ions other than zinc ion.
  • FIG. 3 shows spectral characteristics of the compound of the present invention.
  • FIG. 4 shows spectral characteristics of the compound of the present invention. a) Fluorescence spectra of Compound (15) with excitation wavelengths fixed at 325 nm, b) Excitation spectra of Compound (15) with fluorescence wavelengths fixed at 445 nm.
  • FIG. 5 shows changes in concentration of intracellular zinc ions when Compound (13) is used, which are shown as changes in the ratios of fluorescence intensity on excitations at 340 nm and 380 nm with fluorescent microscope.
  • Arrow (1) 150 ⁇ M of zinc sulfate and 15 ⁇ M of pyrithione were added, and at the time point of Arrow (2), 400 ⁇ M of TPEN was added
  • FIG. 6 shows image of transmitted light through cells and changes in fluorescence intensity ratio when Compound (13) was used.
  • (a) shows a result of transmitted light image
  • (b) shows a result before stimulation
  • (c) shows a result after increasing concentration of intracellular zinc ions by using zinc sulfate and pyrithione
  • (d) shows a result after lowering concentration of intracellular zinc ions by using TPEN.
  • alkyl group or an alkyl moiety of a substituent containing the alkyl moiety (for example, an alkoxy group) used in the specification means, for example, a linear, branched, or cyclic alkyl group, or an alkyl group comprising a combination thereof having 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. More specifically, a lower alkyl group (an alkyl group having 1 to 6 carbon atoms) is preferred as the alkyl group.
  • Examples of the lower alkyl groups include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, cyclopropylmethyl group, n-pentyl group, and n-hexyl group.
  • Methyl group is preferable as the alkyl group represented by R 1
  • methoxy group is preferable as the alkoxy group represented by R 1 .
  • Methoxy group is particularly preferable.
  • methyl group is preferable as the alkyl group represented by X 1 , X 2 , X 3 , and X 4 in R 2 .
  • aryl group in the carboxy substituted aryl group represented by R 3 a monocyclic or condensed aryl group may be used.
  • phenyl group, naphthyl group, and the like are preferable. Phenyl group is particularly preferable.
  • a heteroaryl group in the carboxy substituted heteroaryl group represented by R 3 a monocyclic or condensed heteroaryl group may be used.
  • the number and the type of hetero atom in the heteroaryl group are not particularly limited.
  • the heteroaryl group may contain 1 to 4, preferably 1 to 3, more preferably 1 or 2 hetero atoms selected from the group consisting of nitrogen atom, oxygen atom, and sulfur atom.
  • Oxazolyl group is preferable as the heteroaryl group.
  • the number of the carboxy groups substituting on the aryl ring or the heteroaryl ring is not particularly limited, and preferably 1 to 2, most preferably 1.
  • the carboxy group substituting on the aryl ring or the heteroaryl ring may have a protective group.
  • the protective groups for carboxyl groups “Protective Groups in Organic Synthesis,” (T. W. Greene, John Wiley & Sons, Inc. (1981)) and the like can be referred to.
  • the carboxyl group having a protective group include esters, particularly alkoxycarbonyl groups.
  • a particularly preferable example of the alkoxycarbonyl group includes methoxycarbonyl group.
  • acetoxymethyl group is used as the protective group of the carboxyl group so that the carboxyl group having the protective group is acetoxymethyloxycarbonyl group.
  • the position of the carboxyl group which substitutes on the aryl ring or the heteroaryl ring is not particularly limited, and para-position is preferable when phenyl group is used as an aryl group.
  • both of X 1 and X 2 are preferably 2-pyridylmethyl groups, and further, when m is 1, X 3 is preferred to be hydrogen atom.
  • Y represents a single bond or —CO—, and Y is preferred to be a single bond.
  • R 3 is preferred to be a carboxy-substituted aryl group or a carboxy-substituted heteroaryl group, more specifically, R 3 is preferred to be a carboxy-substituted phenyl group or a carboxy-substituted oxazolyl group.
  • the compounds of the present invention represented by the general formula (I) can exist as acid addition salts or base addition salts.
  • the acid addition salts include: mineral acid salts such as hydrochloride, sulfate, and nitrate; and organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate.
  • the base addition salts include: metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts; ammonium salts; and organic amine salts such as triethylamine salts.
  • salts of amino acids such as glycine may be formed.
  • the compounds or salts thereof according to the present invention may exist as hydrates or solvates, and these substances fall within the scope of the present invention.
  • the compounds of the present invention represented by the aforementioned formula (I) may have one or more asymmetric carbons depending on the types of the substituents.
  • Stereoisomers such as optically active substances based on one or more asymmetric carbons and diastereoisomers based on two or more asymmetric carbons, as well as any mixtures of the stereoisomers, racemates and the like fall within the scope of the present invention.
  • the compounds of the present invention represented by the aforementioned formula (I) or salts thereof are useful as fluorescent probes for zinc.
  • the compounds of the present invention represented by the aforementioned formula (I) or salts thereof will give a remarkable wavelength shift of a peak in an excitation spectrum, when they forms zinc complexes by trapping zinc ions.
  • the wavelength shift can be observed in general as much as about 20 nm width depending on the concentration of zinc ions.
  • the shift can also be observed as a wavelength shift specific to a zinc ion without influence of other metal ions (for example, sodium ions, calcium ions, potassium ions, or magnesium ions).
  • the compound of the present invention as a fluorescent probe for zinc; and by selecting two appropriately different wavelengths to carry out excitation and measuring a ratio of the fluorescence intensities at the excitations, zinc ions can be measured by the ratio method.
  • the two different wavelengths may be selected in such a manner that fluorescence intensity increases with increase of zinc ion concentration at one wavelength, whilst fluorescence intensity decreases with the increase of zinc ion concentration at the other wavelength.
  • the details of the ratio method are described in the book by Mason W. T. (Mason W. T. in Fluorescent and Luminescent Probes for Biological Activity, Second Edition, Edited by Mason W. T., Academic Press). Specific examples of the measurement method using the compounds of the present invention are also shown in Examples of the specification.
  • the compounds of the present invention represented by the aforementioned general formula (I) or salts thereof are featured that they can specifically trap zinc ions and form complexes very rapidly. Accordingly, the compounds of the present invention represented by the aforementioned formula (I) or salts thereof are very useful as fluorescent probes for zinc for measurement of zinc ions in living cells or living tissues under a physiological condition.
  • the term “measurement” used in the specification should be construed in its broadest sense, including quantitative and qualitative measurements.
  • a method for use of the fluorescent probe for zinc according to the present invention is not particularly limited, and the probe can be used in a similar manner to that of conventional zinc probes.
  • a substance selected from the group consisting of the compounds represented by the aforementioned general formula (I) and salts thereof is dissolved in an aqueous medium such as physiological saline or a buffered solution, or in a mixture of the aqueous medium and a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethylsulfoxide, and dimethylformamide, and then the resultant solution is added to a suitable buffered solution containing cells or tissues.
  • the solution is excited at suitably selected two wavelengths, and each of fluorescence intensities may be measured.
  • Compound 11 shown in the above scheme has the excitation wavelength at 354 nm, and the fluorescence wavelength at 532 nm.
  • the compound is used at 20 ⁇ M as a fluorescent probe for zinc, zinc ions at a concentration of about 20 ⁇ M or below are trapped, and as a result, a peak of an excitation spectrum will give about 20 nm blue shift depending on the concentration of zinc ions. Therefore, when this compound is used as a probe, excitation wavelengths such as 335 nm and 354 nm may be used, and the fluorescence intensity at each excitation wavelength is measured to calculate the ratio.
  • the fluorescent probe for zinc according to the present invention may be combined with a suitable additive to use in the form of a composition.
  • the fluorescent probe for zinc can be combined with additives such as a buffering agent, a solubilizing agent, and a pH modifier.
  • Compound (2) was prepared according to the method described in Journal of Organometalic Chemistry, 2000, 611, pp.586-592.
  • Compound (2) (4.7 g: 29 mmol) was dissolved in 150 ml of ethanol, added with 12 g (0.12 mol) of sodium carbonate and 9.6 g (58 mmol) of 2-(chloromethyl) pyridine hydrochloride, and heated under reflux for one day.
  • Ethanol was evaporated under reduced pressure, and the residue was suspended in a 2N aqueous sodium hydroxide solution, which was then extracted with dichloromethane.
  • the organic layer was washed with saturated brine, and dried over potassium carbonate.
  • the dichloromethane was evaporated under reduced pressure.
  • the residue was purified by alumina column to obtain 9.9 g of Compound (3) (yield: quantitative).
  • the reaction mixture was neutralized with 2N hydrochloric acid, and the solvent was evaporated under reduced pressure.
  • the residue was purified by reversed phase HPLC to obtain 20 mg (40 nmol) of Compound (14). Yellow oil. Yield 15%.
  • the compound was precipitated as hydrochloride, and used for measurements.
  • the reaction mixture was neutralized with 2N hydrochloric acid, and the solvent was evaporated under reduced pressure.
  • the residue was purified by reversed phase HPLC to obtain 2 mg (4.4 nmol) of Compound (14). Yellow oil. Yield 1.6%.
  • the compound was precipitated as hydrochloride, and used for measurements.
  • FIG. 4 shows a) Fluorescence spectra of Compound (15) with excitation wavelengths fixed at 325 nm, and b) excitation spectra of Compound (15) with fluorescence wavelengths fixed at 445 nm.
  • FIG. 6 shows image of transmitted light through cells and changes in fluorescence intensity ratio which are pictured using pseudo-color technique.
  • the compound of the present invention is useful as a fluorescent probe for measurement of zinc.
  • the compound of the present invention will give a wavelength shift of a peak in an excitation spectrum after formation of a complex with a zinc ion, and therefore, the zinc ions can be precisely measured by the ratio method by using different two excitation wavelengths.
  • influences of a concentration of fluorescent probe, per se, and intensities of excitation light are negligible, and measurement errors due to localization, concentration change, and discoloration of fluorescent probe per se can be eliminated. Accordingly, the compound of the present invention is extremely useful as a probe for a precise measurement of zinc ions in living organisms.

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PCT/JP2002/005900 WO2002102795A1 (fr) 2001-06-14 2002-06-13 Sondes fluorescentes pour le zinc

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Cited By (2)

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US20070298507A1 (en) * 2004-02-23 2007-12-27 Tetsuo Nagano Fluorescent Probe
US20110117666A1 (en) * 2007-03-01 2011-05-19 The University Of Tokyo Fluorescent probe

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EP1260510B1 (de) * 2000-02-28 2006-10-18 Daiichi Pure Chemicals Co., Ltd. Fluoreszierende proben zur quantitativen bestimmung von zink
KR20030031468A (ko) * 2000-02-29 2003-04-21 다이이치 가가쿠 야쿠힝 가부시키가이샤 활성산소 측정용 시약

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070298507A1 (en) * 2004-02-23 2007-12-27 Tetsuo Nagano Fluorescent Probe
US7939330B2 (en) 2004-02-23 2011-05-10 Tetsuo Nagano Fluorescent probe
US20110117666A1 (en) * 2007-03-01 2011-05-19 The University Of Tokyo Fluorescent probe
US8465985B2 (en) 2007-03-01 2013-06-18 The University Of Tokyo Fluorescent probe

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JP4309253B2 (ja) 2009-08-05
WO2002102795A1 (fr) 2002-12-27
EP1403268A1 (de) 2004-03-31
JPWO2002102795A1 (ja) 2004-09-30
EP1403268A4 (de) 2004-11-24
US20080261314A1 (en) 2008-10-23

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